Lyme disease (LD), caused by the spirochete Borrelia burgdorferi (Bb), is the most common arthropod-borne infection in the United States. Bb is maintained in nature via an enzootic cycle that typically involves rodents and Ixodes ticks. To sustain this complex life cycle, Bb must continuously alter its transcriptome, proteome, and metabolome in response to arthropod- and mammalian host-derived signals. Our long term objective has been to characterize the environmental cues, regulatory pathways, and genes critical for these adaptive changes. In our previously funded application, we hypothesized that the alternative sigma factor RpoS acts as a gatekeeper that (i) transcribes genes required for tick transmission as well as the establishment of mammalian infection and (ii) represses tick-phase genes during mammalian host-adaptation. Our validation of this hypothesis has yielded powerful new insights into the mechanisms that perpetuate the bacterium in nature and maintain Lyme disease as a global threat to human health. A number of our recent findings relate to the transmission of spirochetes during nymphal feeding, the seminal event for infection of humans as well as mice. Using methodologies developed for tracking and imaging live spirochetes within ticks, we discovered that dissemination of Bb in feeding nymphs involves an initial non-motile phase (termed adherence-mediated migration) followed by a transition to motility that enables individual organisms to penetrate the midgut. These studies have helped elucidate the range of adaptations, physiological as well as structural, that spirochetes must undergo in order to negotiate the formidable barriers they encounter en route to the mammal. They also have definitively demonstrated that RpoS-regulated genes are required for transmission of Bb (see Progress Report). Our collective results give rise to our primary hypothesis, to be assessed in Aims 1 and 2, that the RpoS regulon contains many uncharacterized genes that promote the biphasic dissemination of spirochetes and that RpoS-mediated changes in the borrelial transcriptome collectively comprise a 'Go' signal for tick-to-mammal transmission. Additionally, we and others have identified a global regulatory network controlled by the Hk1/Rrp1 two-component system, which we conceptualize as imparting a tick- adaptive 'Stay' signal via the small nucleotide messenger cyclic di-GMP, a molecule known to promote sessile developmental states in bacteria. Our finding that the Hk1/Rrp1 regulon includes tick-phase genes repressed by RpoS within the mammal, coupled with our live-imaging studies, leads to our secondary hypothesis, to be assessed in Aim 3, that the interplay between RpoS and the Hk1/Rrp1 pathway distinguishes spirochete behavior during transmission from acquisition. The underlying premise of these studies is that improved understanding of the molecular mechanisms whereby Bb is transmitted by the arthropod vector will lead to new prevention strategies.